Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4711—Alzheimer's disease; Amyloid plaque core protein
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
  • B01J20/20—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising free carbon; comprising carbon obtained by carbonising processes
  • B01J20/205—Carbon nanostructures, e.g. nanotubes, nanohorns, nanocones, nanoballs
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B82—NANOTECHNOLOGY
  • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
  • B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B82—NANOTECHNOLOGY
  • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
  • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B82—NANOTECHNOLOGY
  • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
  • B82Y35/00—Methods or apparatus for measurement or analysis of nanostructures
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B82—NANOTECHNOLOGY
  • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
  • B82Y40/00—Manufacture or treatment of nanostructures
• • C—CHEMISTRY; METALLURGY
  • C01—INORGANIC CHEMISTRY
  • C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
  • C01B32/00—Carbon; Compounds thereof
  • C01B32/15—Nano-sized carbon materials
  • C01B32/18—Nanoonions; Nanoscrolls; Nanohorns; Nanocones; Nanowalls
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
  • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01Q—SCANNING-PROBE TECHNIQUES OR APPARATUS; APPLICATIONS OF SCANNING-PROBE TECHNIQUES, e.g. SCANNING PROBE MICROSCOPY [SPM]
  • G01Q60/00—Particular types of SPM [Scanning Probe Microscopy] or microscopes; Essential components thereof
  • G01Q60/24—AFM [Atomic Force Microscopy] or apparatus therefor, e.g. AFM probes
  • G01Q60/38—Probes, their manufacture, or their related instrumentation, e.g. holders
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01Q—SCANNING-PROBE TECHNIQUES OR APPARATUS; APPLICATIONS OF SCANNING-PROBE TECHNIQUES, e.g. SCANNING PROBE MICROSCOPY [SPM]
  • G01Q70/00—General aspects of SPM probes, their manufacture or their related instrumentation, insofar as they are not specially adapted to a single SPM technique covered by group G01Q60/00
  • G01Q70/08—Probe characteristics
  • G01Q70/10—Shape or taper
  • G01Q70/12—Nanotube tips
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
  • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
  • G01N2333/4701—Details
  • G01N2333/4709—Amyloid plaque core protein
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2500/00—Screening for compounds of potential therapeutic value
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/28—Neurological disorders
  • G01N2800/2814—Dementia; Cognitive disorders
  • G01N2800/2821—Alzheimer

Definitions

• This invention pertains to the field of high throughput screening.
• a screening system comprising a reconstituted amyloid beta protein channel is disclosed.
• Alzheimer's, Huntington's, Parkinson's, prion encephalopathies, as well as familial British and Danish dementias (FBD, FDD)), systemic (e.g., type II diabetes, light chain amyloidosis) and other (e.g., cystic fibrosis) diseases result from protein misfolding that alters the three-dimensionsl (3D) conformation of the protein from native (often soluble) to non-native (often insoluble) folded structures ⁇ see, e.g., Temussi et al.
• amyloid fibrillar features termed amyloid that results in a gain-offunction and induces a pathophysiological cellular response by altering cell membrane composition and destabilizing cellular ionic homeostasis.
• amyloidosis fibrillar features termed amyloid that results in a gain-offunction and induces a pathophysiological cellular response by altering cell membrane composition and destabilizing cellular ionic homeostasis.
• a multiple planar patch clamp system has been designed that uses lateral cell trapping junctions to reduce capacitive coupling and allows for multiplexed parallel patch sites (Seo et al. (2004) Appl. Phys. Letts., 84: 1973-1975).
• AFM has been successfully used to study the 3D structure of several types of amyloid ion channels related to protein misfolding disease (see, e.g., Example 2, herein, Quist et al. (2005) Proc. Natl. Acad. ScL, USA, 102: 10427-10432; Lin et al. (2001) FASEB J., 15: 2433-2444).
• Example 2 herein, Quist et al. (2005) Proc. Natl. Acad. ScL, USA, 102: 10427-10432; Lin et al. (2001) FASEB J., 15: 2433-2444.
• a direct correlation of the 3D structure and activity of single ion channels is yet to be demonstrated.
• the present invention relates to the discovery of a novel channel structure of human amyloid beta protein (AbP) in lipid membranes and a rapid, quantitative and specific assay for screening test compounds, such as drugs, ligands (natural or synthetic), proteins, peptides and small organic molecules for their ability to bind and block the membrane AbP channels.
• test compounds such as drugs, ligands (natural or synthetic), proteins, peptides and small organic molecules for their ability to bind and block the membrane AbP channels.
• the invention further relates to screening and identifying therapeutically relevant compounds for treating Alzheimer's disease and other disorders.
• this invention provides a device for screening for molecules that alter ion channel activity.
• the device typically comprises a lipid bilayer attached to a solid support, where the lipid bilayer contains one or more ion channel proteins.
• the solid support comprises one or more nanopores (e.g.,
• the nanopores range in size from about 10 nm to about 400 or 500 nm in diameter, preferably from about 20 nm to about 200 nm in diameter, more preferably from about 50 nm to about 100 nm in diameter. In certain embodiments the nanopores range in diameter from about 5 nm, 10 nm, 20 nm, 30 nm, or 50 nm to about 500 nm, 400 nm, 300 nm, 250 nm, 200 nm, 150 nm, 100 nm, 90 nm, 80nm, or 70 nm.
• the nanopores penetrate through a surface having a thickness of about 400 nm or less, preferably about 300 nm or less, and more preferably about 200 nm or less.
• the nanopores are formed in a membrane and/or a silicon wafer and are optionally disposed so that one or more nanopores aligns with an ion channel.
• the device can further comprise a fluid reservoir on one side and/or on the other side of the lipid bilayer.
• the one or more ion channel proteins are selected from a group consisting of a calcium channel, a sodium channel, a potassium channel, a chloride channel, and a magnesium channel.
• the one or more ion channel proteins comprise amyloid proteins ⁇ e.g., AbP channel proteins).
• the device can optionally further comprise a means for detecting alteration of channel conformation in response to contact with a compound.
• a means for detecting alteration of channel conformation in response to contact with a compound include, but are not limited to an atomic force microscope (AFM) probe or a scanning probe microscopy (SPM) probe.
• the device can comprise means to provide a measure of ion channel conductivity.
• the means provides both a measure of channel conductivity and channel protein conformation.
• the means provides a measure of channel conductivity and additionally comprises an AFM or an SPM.
• the device comprises a plurality of different channels ⁇ e.g. at least
• a plurality of the channels are each aligned with a pore in the solid support.
• a method of screening a test agent for the ability to alter conductivity or conformation of an ion channel typically involves contacting a device as described above with a test agent; and detecting a change in conformation and/or conductivity of a channel in response to the contact with the test agent.
• change in conformation is measured using AFM or SPM.
• the change in conductivity is measured using an AFM or SPM tip as an electrode.
• the change in conformation and change in conductivity are measured simultaneously.
• This invention also provides a method of screening test agents for the ability to alter pore conformation or conductance by amyloid proteins.
• the method typically involves providing a lipid bilayer comprising a pore comprising one or more amyloid proteins; contacting the lipid bilayer with a test agent; and detecting a change in the conformation and/or conductance of the pore, where a change in conformation and/or conductance indicates that the test agent alters pore conformation or conductance.
• this invention provides an AFM or SPM having an integrated carbon nanotube cantilever and tip.
• This invention also provides a carbon nanocone, or an AFM or SPM having a carbon nanocone tip.
• the nanocone typically comprises a high-aspect ratio carbon nanotube structure substantially lacking a catalyst at the tip.
• the nanocone has a cone angle of less than about 15degrees, preferably less than about 10 degrees, more preferably less than about 5 degrees.
• the nanocone has an aspect ratio (height:base) of at least about 5:1, preferably lat least about 10:1, more preferably at least about 12:1.
• the nanocone has a tip radius of less than about 10 nm, preferably less than about 5 nm, more preferably less than about 3 nm.
• the method typically comprises a resist-free e-beam induced deposition (EBID) of carbon masks combined with electric-field-controlled CVD growth.
• EBID resist-free e-beam induced deposition
• the method utilizes EBID carbon patterns as dry etching masks.
• Ion channels are proteins in cell membranes that act as pores to permit the passage of charged species (ions) across the cell membrane ⁇ e.g., a lipid bilayer).
• the ion channels have the ability to open or close in response to specific stimuli and thus allow for gating of ions in and out of enclosed subcellular compartments and/or whole cells.
• Ion channel proteins can be referred to by the type of ion they pass.
• a calcium channel is an ion channel that selectively or preferentially allows the passage of calcium ion (Ca 2+ ) through a membrane.
• An ion channel protein is a protein that is a component of an ion channel.
• test agent refers to an agent that is to be screened in one or more of the assays described herein.
• the agent can be virtually any chemical compound. It can exist as a single isolated compound or can be a member of a chemical (e.g. combinatorial) library. In a particularly preferred embodiment, the test agent will be a small organic molecule.
• small organic molecule refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals.
• Preferred small organic molecules range in size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da.
• Figure 1 shows CD spectra of mutant amyloid molecules in solution.
• CD spectrometry analysis of ABri, ADan, ⁇ -synuclein, amylin, SAA, and A ⁇ (l-40) in 5 mM Tris (pH 7.4) was carried out on a Jasco J-720 spectropolarimeter at 1-nm intervals over the wavelength range 190-260 nm at 24°C in a 0.1-cm path-length cell. Results are expressed in molar ellipticity (deg cm 2 mol "1 -).
• Figure 2 shows Electrophoresis of ABri, ADan, ⁇ -synuclein, amylin, SAA, and A ⁇ (l- 40) on 16.5% SDS-PAGE under reducing conditions.
• the right lane shows peptide in aqueous solution; the left lane shows peptide in DOPC membrane after solubilization in 2% SDS. Positions of molecular mass markers are indicated on the left.
• Amylin and A ⁇ (l-40) were cross-linked both in solution and in membrane. In solution, for all peptides the monomers are observed. Small amounts of dimers are observed in solution for A ⁇ (l-40), ABri, and amylin.
• FIG. 3 shows single-channel records of amyloid channels. Current traces as a function of time under voltage-clamp conditions are shown.
• Figures 4 shows AFM images of freshly dissolved peptide molecules.
• Green arrows indicate surface-adsorbed peptides with a width of 8-12 nm and a height of 1-2 nm (consistent with the size of monomers). Arrows indicate higher-order oligomers and clusters. For ABri, most observed features are monomers and dimers; for other peptides, larger multimers and aggregates are observed. For ADan and amylin, the amount of monomers adsorbed is small, and mostly multimers and clusters are observed. In one experiment for oc-synuclein, fiber-like structures were observed, indicated by dotted lines.
• Figure 5 shows AFM images of amyloid peptides reconstituted in membrane bilayers. / « « ⁇ shows lipid bilayer with thickness of ⁇ 5 nm.
• a ⁇ (l-40) ADan
• ⁇ - synuclein channel-like structures with a central pore can be easily resolved.
• SAA SAA
• amylin the central pore is only resolved on some multimer structures. Arrows indicate locations where annular structures can be observed clearly.
• channel sizes are 16 nm for A ⁇ (l-40), 14 nm for ABri, 14 nm for ADan, 16 nm for ⁇ -synuclein, 12 nm for SAA, and 15 nm for amylin. (Scale bars: 100 nm.)
• Figure 6 shows individual channel-like structures at high resolution. Two examples are shown for each molecule, in which a central pore can be observed. The number of subunits observed protruding from the surface varies from four to eight subunits. Resolution of AFM images is not enough to resolve individual subunit structures. [Image sizes are 25 nm forA ⁇ (l-40), 25 nm for ⁇ -synuclein, 35 nm for ABri, 20 nm for ADan, 25 nm for amylin, and 20 nm for SAA.] [0027] Figure 7 schematically illustrates the cross-section of a conventional probe.
• FIG 8 panels A, B, and C illustrate processing steps for a probe as described herein.
• Figure 9 illustrates a schematic of a probe assembly with a detection mechanism.
• Figure 10 illustrates a vertically grown nanotube.
• Figure 11 illustrates a vertically grown nanotube with an added CNT segment grown at an angle.
• Figure 14 illustrates examples of carbon nanotube growth manipulation.
• FIG. 15 Panel A: Tapping mode AFM image of arrays of nanopores (left).
• the pores are produced in the silicon nitride windows using electron beam lithography and range in size from 50 to 100 nm.
• Panel B After deposition of lipid vesicles, a bilayer is formed covering the pores (center, imaged under buffer). The bilayer has several holes; the cross section on a hole in the bilayer shows a depth of 5.5 nm, the typical thickness of a lipid bilayer. Scale bars 1 micron.
• Panel C shows a schematic of the liquid cell AFM setup imaging the nitride window. [0036]
• Figure 16 shows force distance curves acquired over an alignment mark that is partially covered by a lipid bilayer.
• Crosses indicate position of force curves acquired on the substrate (top), edge of the mark (middle) and a bilayer suspended over the mark (bottom).
• the increased z-distance from point of contact to pre-set deflection indicates a softer sample (arrows).
• the inset shows a stable suspended bilayer imaged repetitively using 1.5 nN force. Scale bar 500 nm.
• Figure 17A shows combined AFM height and current imaging.
• the left images show topography of the same FBB milled pore imaged repeatedly with varying bias potential, -2.0 V (top), -1.0 V (middle), and -0.5 V (bottom).
• the right images are current images, a distinct current signal is observed over the pore.
• the amount of current is shown in the cross sections of the current images, 214 pA at -2.0 V, 65 pA at -1.0 V, and 5 pA at - 0.5 V.
• Figure 17B shows a schematic setup with the platinum electrode in buffer under the nitride window. Scale bar 1 micron.
• Figure 18A shows IV curves collected 10 seconds (top), 2 minutes (middle) and 4 minutes (bottom) after addition of Gramicidin to the lipid bilayer sample suspended over the nanopore. IV voltage range -0.5 to 0.5 Volt. Conductance increases from 0.025 nS (bilayer only) to 0.25 nS, 0.5 nS and 1 nS respectively.
• Figure 18B shows a schematic setup
• Figure 18C shows a schematic of the FIB induced nanopore with deposited oxide and bilayer.
• panels A-F schematically illustrates a fabrication procedure.
• Panel A Device layer (gray), buried oxide layer (cross-hatched) and handle wafer (gray).
• F Nanopore created by FEB, diameter 70 nm.
• Figure 20 schematically illustrates a setup for AFM and combined electrical recording. Images of topography and current are acquired simultaneously.
• FIG. 21 panels A-D, show a schematic of the resist-free fabrication technique for a single CNC based AFM tip.
• Panel A Catalyst deposition by e-beam evaporation.
• Panel B E-beam induced deposition of a carbon dot mask.
• Panel C Metal wet etching and the removal of a carbon dot.
• Panel D CVD growth of a CNC probe.
• FIG. 22 Panel A-D, show schematics and SEM of carbon nanotubes and nanocone formation.
• Panel A Schematic of an equidiameter carbon nanotube growth.
• Panel B SEM image of equidiameter CNTs grown on an Si substrate at a dc bias of 450 V for 20 min.
• Panel C Schematic of the gradual reduction of a catalyst particle at the CNT tip by sputtering and accompanying reduction in the CNT diameter.
• Panel D SEM image of the CNC with the catalyst particle completely removed at 550 V for 20 min.
• FIG. 23 panels A and B, show an SEM image of a single CNC probe.
• Panel A Top view SEM image of the single CNC probe grown near the edge of a cantilever (low-magnification, 30° tilted view).
• Panel B Side view SEM image of the CNC probe.
• Inset TEM image of the CNC tip.
• panels A-F shows a comparison of AFM imaging using a nanocone and a conventional tip.
• Panel A Copper film AFM image.
• Panel B Zoom-in Cu film AFM image.
• Panel C AFM image of a PMMA line pattern by a conventional Si pyramid tip.
• Panel D Image of the same PMMA pattern by a CNC tip.
• Panel E The height profile of image panel C.
• Panel F The height profile of image panel D.
• This invention pertains to the discovery that certain protein misfoldings, in particular the misfolding of various channel proteins (e.g., ion channel proteins) is implicated in the etiology of various pathologies.
• Protein conformational diseases including, but not limited to Alzheimer's disease, Huntington's disease, Parkinson's disease, and the like, result from protein misfolding, giving a distinct fibrillar feature termed amyloid.
• Recent studies have shown that only the globular (not fibrillar) conformation of amyloid proteins is sufficient to induce cellular pathophysiology. However, the 3D structural conformations of these globular structures, a key missing link in designing effective prevention and treatment, has remained undefined.
• amyloid molecules including amyloid- ⁇ (l- 40), ⁇ -synuclein, ABri, ADan, serum amyloid A, and amylin undergo supramolecular conformational changes.
• amyloid- ⁇ (l- 40) including amyloid- ⁇ (l- 40), ⁇ -synuclein, ABri, ADan, serum amyloid A, and amylin undergo supramolecular conformational changes.
• they form morphologically compatible ion-channel-like structures and elicit single ion-channel currents. These ion channels would destabilize cellular ionic homeostasis and hence induce cell pathophysiology and degeneration in amyloid diseases.
• ion channels and ion channel proteins particularly those comprising amyloid proteins provide targets to screen for agents that modulate (e.g., inhibit, or stabilize/upregulate) pore formation and such agents are expected to provide effective lead compounds for the development of therapeutics for the treatment of protein conformation pathologies.
• agents that modulate e.g., inhibit, or stabilize/upregulate
• pore formation e.g., pore formation
• agents are expected to provide effective lead compounds for the development of therapeutics for the treatment of protein conformation pathologies.
• the fibril formation of amyloid beta protein (AbP) is not required for AbP-induced cellular toxicity and the non-fibrillar form of AbP, at physiological levels, can induced cell degeneration. Moreover this damage is not via the mechanisms of oxidative damage or binding of tachykinin receptors as previously proposed.
• the present invention relates to rapid, quantitative and specific assays for screening test compounds, such as drugs, ligands (natural or synthetic), proteins, peptides and small organic molecules for their ability to bind and block, or alternatively, in certain cases to stabilize, the membrane ion channels comprising one or more amyloid proteins (e.g., AbP channels).
• test compounds such as drugs, ligands (natural or synthetic), proteins, peptides and small organic molecules for their ability to bind and block, or alternatively, in certain cases to stabilize, the membrane ion channels comprising one or more amyloid proteins (e.g., AbP channels).
• modulation of AbP channels will prevent cellular calcium imbalance and thereby prevent or mitigate symptoms of Alzheimer's disease
• the present invention also relates to the drugs, ligands, proteins, peptides and small organic molecules identified by the screening assay of the present invention as capable of inhibiting membrane AbP channels.
• the invention is based, in part, on our discovery and demonstration that AbP form channel-like structure in lipid membranes. We have seen two specific three dimensional forms of the channel, hexameric and tetrameric channels (Figure 1), which is also consistent with biochemistry assay ( Figure 2) Design of the assay.
• this invention contemplates assays and devices that use liposomes or planar lipid bilayers with incorporated ion channel proteins ⁇ e.g., AbP channels) as target to screen for therapeutically relevant molecules for treating Alzheimer's disease and other disorders.
• the AbP channel proteins include, but are not limited to all peptide channels from by proteolytic product of ⁇ -amyloid precursor protein (AbPP), such as AbP 1-25, AbP 1-39, AbP 1-40, AbP 1-42 , AbP 1-43 , and the like.
• one or more test agents are screened for their ability to bind, preferably to specifically bind, one or more ion channel proteins, preferably amyloid ion channel proteins when present in a lipid bilayer.
• the amyloid ion channel proteins can be introduced into an isolated bilayer ⁇ e.g. a bilayer attached to a solid support), into a liposome comprising a bilayer, into an oocytes comprising a bilayer ⁇ e.g., a Xenopus oocytes), into a cell, and the like.
• Binding of the test agent(s) to the amyloid channel protein(s) can be detected by any of a number of methods known to those of skill in the art.
• the test agent(s) are labeled with a detectable label ⁇ e.g., a fluorescent label, a colorimetric label, a radioactive label, a spin (spin resonance) label, a radioopaque label, etc.).
• the membrane comprising the amyloid channel protein(s) is contacted with the test agent(s), typically washed, and then the membrane is screened for the detectable lable indicating association of the test agent with the amyloid channel protein.
• a secondary binding moiety ⁇ e.g.
• the label on the test agent and the label on the secondary agent can be labels selected that undergo fluorescent resonance energy transfer (FRET) so that excitation of one label results in emission from the second label thereby providing an efficient means of detecting association of the labels.
• FRET fluorescent resonance energy transfer
• the assay is a competitive assay format.
• a "competitive" agent e.g., antibody, small organic molecule, etc.
• the competitive agent can be labeled and the amount of such agent displaced when the bilayer containing the amyloid protein(s) is contacted with a test agent provides a measure of the biding of the test agent.
• Methods of detecting specific binding are well known and commonly used, e.g. in various immunoassays. Any of a number of well recognized immunological binding assays (see, e.g., U.S.
• Patents 4,366,241; 4,376,110; 4,517,288; and 4,837,168 are well suited to detection of test agent binding to amyloid channel proteins in a lipid bilayer.
• binding of test agents can be detected by detecting alterations (e.g., decrease) of ion (e.g., Ca 2+ ) uptake by a cell, oocytes, or liposome in the presence of the test agent(s)-uptake in to the liposomes.
• ion e.g., Ca 2+
• This can readily be detected using for example a radio-isotope (e.g., 45 Ca 2+ ) or a calcium sensitive dye (e.g., arsenazo El (AIII), Fura-2, Fluo-3, Fluo-4, Calcium green, etc.).
• the modulation of amyloid channels by test agent(s) can be detected by monitoring changes in ionic channel conductances in cells or oocytes, or in liposomes, lipid layers, or other ex vivo systems.
• Methods of detecting ion channel conductivity are well known to those of skill in the art.
• single channel ion currents are studied using the patch-clamp technique (see, e.g., Neher and Sakmann (1976) Nature, 260: 799-802; Sakmann and Neher (1983) Single Channel Recording, Plenum New York), in which a glass pipette filled with electrolyte is used to contact the membrane surface and measure ionic current.
• Various chip-based patch clamping methods are also known (see, e.g., Fertig et al. (2002) Appl. Phys. Letts., 81: 4865-4867).
• this invention contemplate the use of an ex vivo system comprising two chambers separated by a lipid bilayer, that contains an amyloid protein ion channel. The conductance across the lipid bilayer is monitor continuously.
• This device can be used to assay molecules that block or modulate the activity of, for example, AbP channels. Various features of such a device are described in more detail below.
• alterations of channel conductivity and/or conformation can be measured using scanning probe microscopy (SPM) and/or atomic force microscopy (AFM).
• SPM scanning probe microscopy
• AFM atomic force microscopy
• Chip-based SPM/AFM devices for screening channel proteins conformation changes are Chip-based SPM/AFM devices for screening channel proteins conformation changes.
• this invention contemplates chip-based supported bilayer systems for screening test agents for their ability alter ion channel protein conformation and/or conductance.
• the device comprises a lipid bilayer attached to (disposed on) a support.
• the support is typically a microfabricated support (e.g., micromachined using photolithographic methods and/or various ion beam etching methods).
• the support comprises one or more nanopores.
• the nanopores typically range in size from about 10 nm to about 400 nm, preferalbly from about 20 nm to about 200 nm in diameter, more preferably from about 50 to about 100 nm in diameter.
• the nanopores range from about 10 nm, 20nm, 30 nm, 50 nm, or 70 nm in diameter, to about 400 nm, 300 nm, 250 nm, 200 nm, 150 nm, or 100 nm in diameter.
• Support can be a rigid support, or in certain embodiments, a membrane support.
• the nanopores can be produced in electron beam lithography as well as by using a finely focused ion beam.
• Thermal oxide can used to shrink pore sizes, if necessary and to create an insulating surface.
• the chips with well defined pores can be mounted on a double chamber plastic cell recording system allowing for controlling the buffer conditions both above and below the window (membrane).
• the system can be oriented to permit use of an AFM or SPM tip to measure ion channel protein conformation.
• the AFM or SPM tip can also function as an electrode and with a second electrode (e.g., a platinum wire) under the membrane window conductance across lipid bilayers that are suspended over the pores can readily be measured.
• the probes can, optionally, further comprise a nanotubes that functions as an electrode.
• the fabrication and use of such a system is illustrated herein in Example 3. Using the teaching provided herein, other variants of such systems will readily be available to one of skill in the art.
• this invention utilizes microfabricated SPM or AFM probes preferably having an integrated carbon nanotube cantilever and tip.
• SPMs scanning probe microscopes
• the tip is coated with different materials to perform measurements such as electrical, magnetic, etc. Obtaining a very high resolution images has always been a goal for the scientific community. Since silicon is very brittle, the sharp tips do not last very long on hard surfaces. On the other hand the soft samples such as living cells have risk of getting damaged by the hard tip.
• this invention provides methods of manufacturing
• SPM/ AFM probes that can, optionally, have, both, cantilever and tips made out of carbon nanotubes or similar materials.
• the process of manufacturing, presented in this invention, as shown in the attached figures, utilizes nano-fabrication technology in conjunction with the carbon nanotube growth process.
• the following features of these probes make them unique in their performance: They have a very low spring constant; they have a low squeeze film damping effect in air; they are ideal for imaging in liquid, they have a sharp tip, and a long lifetime.
• Figure 7 shows cross-sectional view of a conventional SPM probe with integrated cantilever and tip.
• the probe has three parts: a substrate, a cantilever and a tip.
• the performance and the applications of a given probe are determined by the cantilever and the tip.
• the substrate facilitates handling and mounting of the probe assembly in the scanning probe microscopes.
• the methods of this invention involve growing a thick and vertical carbon nanotube (CNT) out of the silicon surface and using it as a cantilever.
• the tip made out of CNT also, can be attached at the end of the cantilever.
• a CNT reflector can then be grown near the end of CNT cantilever to facilitate imaging the surface through laser detection.
• the fabrication of silicon substrate and CNT cantilever growth is a batch fabrication process. The following sections briefly describe the process and variations of fabricating the device.
• Figure 8 panel A shows the cross-sectional view of the silicon substrate formed after series of standard microfabrication processing steps mentioned in the diagram.
• the multiple substrates formed in the process can be separated after the carbon nanotube 9 growth as shown in Figure 8, panel B.
• the individual substrates get separated at the deep groove 5 and v-groove 6.
• a gentle touch is sufficient to separate the substrates as they are held by about 20 um thick silicon membrane.
• Figure 8 panel C, shows a cross-sectional view of the completed device with CNT tip and CNT reflector.
• the reflector is coated with a metal layer to make the laser bounce from the source to the detector.
• a schematic of the probe assembly with laser detection system is shown in Figure 9.
• FIG. 10 shows a vertical CNT 9a grown on silicon substrate having a catalyst 8.
• a segment of CNT as a tip is added at an angle at the free end of the CNT cantilever (Figure 11).
• the added segments can be made in different shapes and dimensions for different applications as shown in Figure 12, panels A-D.
• FIG. 13 shows a mounting block for the silicon substrate with catalyst to grow CNT at an angle.
• panels B and C show CNTs grown at an angle in such system.
• the CNT growth angle can be controlled and manipulated with sharp bends to make zig-zag structures leading to spring tips as shown in Figure 14.
• the apex of the CNT tip can be flat or sharp (e.g., 1 - 2 nm).
• the tips described herein have a very low spring constant and can be used, for example in the fabrication of microcantilever arrays for biosensors and the like.
• the CNT cantilevers with sharp tips can be used for deterging changes in pore conductivity or conformation and can also be used for for high resolution images in life science and materials studies.
• the tilted tips are also useful for sidewall roughness measurement.
• the flat apex of the CNT tip will provide reproducibility and long life time.
• the above assays can be implemented in a parallel array for simultaneous screening of multiple different molecules.
• small unilaminar liposomes can be cross-linked to attach onto a solid support, and implanted in a multi-array system, such as a fabricated silicon chip or a multi-well system.
• planar lipid bilayer with incorporated ion channels can be absorbed on a solid support, such as multi-well plates or fabricated chips.
• multiple target compounds can be simultaneously tested, e.g. in a high throughput screening (HTS) format.
• HTS high throughput screening
• high throughput screening methods involve providing a library containing a large number of compounds (candidate compounds) potentially having the desired activity. Such “combinatorial chemical libraries” are then screened in one or more assays, as described herein, to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. The compounds thus identified can serve as conventional "lead compounds" or can themselves be used directly in the desired application.
• A) Combinatorial chemical libraries for modulators ion channel conformation and/or conductivity [0079] The likelihood of an assay identifying an agent that modulates ion channel conformation and/or conductivity is increased when the number and types of test agents used in the screening system is increased. Recently, attention has focused on the use of combinatorial chemical libraries to assist in the generation of new chemical compound leads.
• a combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis by combining a number of chemical "building blocks" such as reagents.
• a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks called amino acids in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks. For example, one commentator has observed that the systematic, combinatorial mixing of 100 interchangeable chemical building blocks results in the theoretical synthesis of 100 million tetrameric compounds or 10 billion pentameric compounds (Gallop et al. (1994) 37(9): 1233-1250). [0080] Preparation and screening of combinatorial chemical libraries is well known to those of skill in the art.
• Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Patent 5,010,175, Furka (1991) Int. J. Pept. Prot. Res., 37: 487-493, Houghton et al (1991) Nature, 354: 84-88).
• Peptide synthesis is by no means the only approach envisioned and intended for use with the present invention.
• Other chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to: peptoids (PCT Publication No WO 91/19735, 26 Dec. 1991), encoded peptides (PCT Publication WO 93/20242, 14 Oct.
• Patent 5,539,083) antibody libraries see, e.g., Vaughn et al. (1996) Nature Biotechnology, 14(3): 309-314), and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al (1996) Science, 21 A: 1520-1522, and U.S. Patent 5,593,853), and small organic molecule libraries (see, e.g., benzodiazepines, Baum (1993) C&EN, Jan 18, page 33, isoprenoids U.S. Patent 5,569,588, thiazolidinones and metathiazanones U.S. Patent 5,549,974, pyrrolidines U.S. Patents 5;525,735 and 5,519,134, morpholino compounds U.S. Patent 5,506,337, benzodiazepines 5,288,514, and the like).
• antibody libraries see, e.g., Vaughn et al. (1996) Nature Biotechnology, 14(3): 309
• a number of well known robotic systems have also been developed for solution phase chemistries. These systems include automated workstations like the automated synthesis apparatus developed by Takeda Chemical Industries, LTD. (Osaka, Japan) and many robotic systems utilizing robotic arms (Zymate II, Zymark Corporation, Hopkinton, Mass.; Orca, Hewlett-Packard, Palo Alto, Calif.) which mimic the manual synthetic operations performed by a chemist. Any of the above devices are suitable for use with the present invention. The nature and implementation of modifications to these devices (if any) so that they can operate as discussed herein will be apparent to persons skilled in the relevant art.
• any of the assays for agents that modulate ion channel conformation and/or conductivity described herein are amenable to high throughput screening.
• Binding assays for example, are well known and U.S. Patent 5,559,410 discloses high throughput screening methods for protein binding, while U.S. Patents 5,576,220 and 5,541,061 disclose high throughput methods of screening for ligand/antibody binding.
• Robotics systems for manipulating reagents and the like in conjunction with such assays are are commercially available (see, e.g., Zymark Corp., Hopkinton, MA; Air Technical Industries, Mentor, OH; Beckman Instruments, Inc. Fullerton, CA; Precision Systems, Inc., Natick, MA, etc.). These systems typically automate entire procedures including all sample and reagent pipetting, liquid dispensing, timed incubations, and final readings of the microplate in detector(s) appropriate for the assay.
• These configurable systems provide high throughput and rapid start up as well as a high degree of flexibility and customization. The manufacturers of such systems provide detailed protocols the various high throughput.
• Zymark Corp. provides technical bulletins describing screening systems for detecting the modulation of gene transcription, ligand binding, and the like.
• Ion channels include, but are not limited to a calcium channel, a sodium channel, a potassium channel, a chloride channel, a magnesium channel, and the like. Protein constituents of various calcium, sodium, potassium, chloride, magnesium channels are known to those of skill. In addition, pathological states attributed to the dysfunction of these channels and particular proteins comprising such channels are also known to those of skill in the art.
• chloride channels include, but are not limited to voltage gated chloride channels (CLC), including, but not limited to CLC-I, CLC-2, CLC-3, CLC-4, CLC-5, CLC-6, CLC-7, CLC-O, ClC-K/barttin channels (e.g.,
• CLC-KA, CLCN-KB chloride intracellular channels CLIC-I, CLIC-2 CLIC-3 CLIC-4 CLIC-5, etc., calcium activated chloride channels (CLACl, CLAC2, CLAC3, etc.), and the like.
• Pathologies associated with dysfunctional chloride channels include but are not limited to myotonia congenita (CLC-I), Myotonic Dystrophy (DMl; DM2), Epilepsy (CLC-2), Renal tubular disorders (CLC-5), Bartter's syndrome (CLC-KB ), cystic fibrosis (epithelial chloride channel ), osteopetrosis, etc.
• Various illustrative sodium channels include, but are not limited to voltage- gated Na + channels, (e.g., SCNl, SCN1ASCN2A1, SCN2A2, SCN3A, SCN4A, SCN5A, SCN7A, SCN8A (PN4), SCN9A (PNl), SCNlOA, SCNIlA, SCNlB ( ⁇ l), SCN2B ( ⁇ 2), SCN3B, SCN4B), non-voltage-gated Na+ channels (e.g., epithelial sodium channel, degennerins, etc.), sodium/hydrogen exchanges (e.g., NAHl, NAH2, NAH3, NAH4, NAH5, SLC9A6, SLC9A7, etc.), SLC5A, SLC24, and the like.
• voltage- gated Na + channels e.g., SCNl, SCN1ASCN2A1, SCN2A2, SCN3A, SCN4A, SCN5A, S
• Pathologies associated with dysfunctional sodium channels include but are not limited to, hyperkalemic periodic paralysis, paramyotonia, myotonia, myasthenia, long qt syndrome 3, progressive cardiac conduction defect (PCCD2; Lenegre-Lev disease), congenital non-progressive heart block, idiopathic ventricular fibrillation, congenital sick sinus syndrome (SCN5A), hyperkalemic periodic paralysis , hypokalemic periodic paralysis , paramyotonia congenita , myotonia fluctuans , myotonia permanens , acetzolamide-responsive myotonia , malignant hyperthermia , myasthenic syndrome, multifocal motor neuropathy, acute motor axonal neuropathy etc.
• PCCD2 progressive cardiac conduction defect
• SCN5A congenital sick sinus syndrome
• hyperkalemic periodic paralysis hypokalemic periodic paralysis
• paramyotonia congenita myotonia fluctuans
• Various illustrative calcium channels include, but are not limited to, voltage- gated Ca ++ channels (e.g., N-type, P-type, L-type, Q-type, R-type, P-type, etc.), ligand-gated Ca ++ channels (e.g., Ca ++ transporting ATPase), capacitive Ca++ entry channels, Intracellular activation channels, calcium sensors, and the like. .
• voltage- gated Ca ++ channels e.g., N-type, P-type, L-type, Q-type, R-type, P-type, etc.
• ligand-gated Ca ++ channels e.g., Ca ++ transporting ATPase
• capacitive Ca++ entry channels e.g., Intracellular activation channels, calcium sensors, and the like.
• Pathologies associated with dysfunctional sodium channels include but are not limited to, hypokalemic periodic paralysis (CACNL1A3 ⁇ lS subunit), malignant hyperthermia (CACNL1A3 ⁇ lS subunit), long QT syndrome with syndactyly (Timothy syndrome), X-linked congenital stationary night blindness, familial hemiplegic migraine, juvenile myoclonic epilepsy, granulomatous myopathy, brody myopathy, Darier- White disease: Keratosis follicularis, etc.
• Various illustrative potassium channels include, but are not limited to, voltage gated potassium channels, inwardly rectifying potassium channels (e.g. (Kir channels, KCNK family, KCNJ family, KCNH family, KCNM family, etc.), delayed rectifier K + channels, Ca ++ sensitive K + channels (e.g. BK, IK, SK), TP-sensitive K + channels, Na + activated K + channels, and the like.
• Pathologies associated with dysfunctional potassium channels include but are not limited to, atrial fibrillation, short QT syndrome, episodic ataxia / myokymia syndrome, myokymia & benign neonatal epilepsy, etc.
• kits for practicing the various methods described herein.
• the kits can include, for example, the assay devices described herein.
• the assay device is a chip based device and is, optionally, provided in a format compatible with a commercially provided reader.
• the reservoirs can, optionally, contain one or more buffers, labels, and/or bioactive agents as required.
• the bioactive agent or other agent is provided in a dry rather than a fluid form so as to increase shelf life.
• the kits can optionally further comprise buffers, syringes, sample collectors and/or other reagents and/or devices to perform one or more of the assays described herein.
• kits typically provided in one or more containers.
• the containers are sterile, or capable of being sterilized (e.g. tolerant of on site sterilization protocols).
• kits can be provided with instructional materials teaching users how to use the device of the kit.
• the instructional materials can provide directions on utilizing the assay device to screen for modulators of ion channels.
• instructional materials typically comprise written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include, but are not limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials.
• electronic storage media e.g., magnetic discs, tapes, cartridges, chips
• optical media e.g., CD ROM
• Such media may include addresses to internet sites that provide such instructional materials.
• FIG. 3 shows an experiment that Zn 2+ and an anti-AbP antibody (3D6) blocks the channels formed by the 40 residue AbP 1-40 and inhibits the uptake of 45 CA 2+ into the liposomes via the AbP channels.
• Figure 4 shows a similar experiments, in which the channels formed by the 42-residue AbP 1-42 was blocked by and antibody, Zn 2+ , and Tris.
• Amyloid Ion Channels A Common Structural Link For Protein-Misfblding Disease
• Protein conformational diseases including Alzheimer's, Huntington's, and
• amyloid a distinct fibrillar feature termed amyloid.
• Recent studies show that only the globular (not fibrillar) conformation of amyloid proteins is sufficient to induce cellular pathophysiology.
• the 3D structural conformations of these globular structures remain undefined as yet.
• an array of amyloid molecules including A ⁇ (l-40), ⁇ -synuclein, ABri, ADan, Serum Amyloid A, and amylin undergo supramolecular conformational change.
• reconstituted membranes In reconstituted membranes, they form morphologically compatible ion-channel-like structures and elicit single ion channel currents. These ion channels would destabilize cellular ionic homeostasis and hence induce cell pathophysiology and degeneration in amyloid diseases.
• DOPC Dioleoyl-OT-glycero-3-phosphocholine
• ⁇ -synuclein recombinant protein ( ⁇ -synuclein; molecular mass, 14.5 kDa) and human apo-serum amyloid A (SAA) were purchased from Alpha Diagnostics (San Antonio, TX) and PeproTech (Rocky Hill, NJ), respectively.
• a ⁇ (l- 40), amylin, ADan, and ABri were synthesized in the W. M. Keck Facility (Yale University) by iV-t-butyloxycarbonyl chemistry and purified by reverse-phase HPLC.
• Tris-N-tris(hydroxymethyl)methylglycine (Tricine)-SDS precast gel cassettes SDS sample buffer, Tris ⁇ Tricine ⁇ SDS running buffer, and molecular mass standards were purchased from Bio-Rad. All solutions were prepared by using ultrapure water (resistivity- 18.2 M—cm-O from Milli-Q from Millipore purification system.
• the pellet was washed by using 10 mM Hepes solution (pH 7.4) and subsequently resuspended in 10 mM Hepes. The procedure was repeated three times to ensure that no unincorporated peptides were left in the mixture. Afterward, liposomes were dissolved in SDS sample buffer (200 mM Tris/HCl/2% SDS/40% glycerol/0.04% Coomassie blue G-250, pH 6.8). SDS sample buffer was added to peptides freshly dissolved in water. The peptides were separated by electrophoresis on 16.5% Tris ⁇ Tricine ⁇ SDS polyacrylamide gels (SDS-PAGE). Molecular mass markers (from Bio-Rad) were run parallel to the samples. Peptides were fixed with 10% acetic acid and stained with Coomassie Blue G-250 (Invitrogen) or silver stain (Bio-Rad).
• Planar lipid bilayers were prepared by means of liposome fusion followed by rupture on the mica surface by procedure modified from Lin et al. (2001) FASEB J., 15: 2433-2444. Briefly, DOPC was dissolved in chloroform and dried under a flow of dry argon. DOPC pellet was vacuum-desiccated over-night and subsequently resuspended in 10 mM Hepes (pH 7.4) to a final concentration of 1 mg/ml. Lipids were hydrated for 1 h during which occasional vortexing was applied. Liposomes then were freeze-thawed and passed subsequently through a set of 400- and 200-nm pore size filters.
• Peptides were dissolved in ultrapure water and mixed with the DOPC liposomes at a 1:20 weight ratio. Lipid-protein mixture was bath-sonicated for 30 sec. Liposomes reconstituted with peptides then were deposited on freshly cleaved mica for 20 min and allowed to fuse and rupture upon contact with the mica surface forming planar lipid bilayers. The sample then was washed, and no additional amyloids were added so that no unincorporated amyloids were left before imaging.
• AFM images were acquired by using Nanoscope Ilia Multimode AFM with an Extender electronics module (Veeco, Santa Barbara, CA) as described in ref. 5.
• Oxide- sharpened silicon nitride cantilevers with a nominal spring constant of ⁇ 0.06 N/m were used for most experiments. Imaging was carried out in both regular contact mode and in tapping mode (at oscillation frequencies between 9 and 15 kHz). Occasionally, higher-frequency resonance peaks (28-33 kHz) were used. The scan rates ranged between 1 and 12 Hz. All imaging was performed in 10 mM Hepes solution (pH 7.4) by using AFM liquid cell at room temperature. Through a continuous adjustment of the scanning parameters, it was ensured that imaging did not affect surface structure by routinely examining for damage by increasing the scan size at regular time intervals.
• AFM images were processed and analyzed by using Veeco software.
• AFM images were low-pass filtered. Single ion channels images were passed through an additional low-pass Gaussian filter to reduce pixilation. Sizes of freshly dissolved peptide molecules as well as reconstituted channels in membrane were obtained by cross- sectional and bearing analyses software. The size of the structures observed in the cross- sections of height mode AFM images were measured at two-thirds of full height with respect to the substrate plane (mica surface for freshly dissolved nonmembrane-associated peptides; the bilayer membrane surface for amyloid channels) (Lin et al. (2001) FASEB J., 15: 2433-2444). Sizes and pore statistics for reconstituted channels were obtained from 50-200 channel-like features for each particle in amplitude-mode images. For the bilayers reconstituted with the peptide, often low gains in AFM imaging were required, rendering the amplitude image more reliable for analysis than the height images. Results
• Single-channel recording could often be seen to merge into macroscopic conductances, implying that the former are responsible for the latter.
• One-sided addition of amyloid peptide to the solution bounding a bilayer membrane was sufficient to induce channel activity. Multiple sizes of single channels usually could be observed that depended on the peptide aggregation state. Channels were never observed in the absence of added peptide. Channel-forming activity could sometimes vary depending on the aggregation state of the peptide; e.g., disaggregation with DMSO followed by brief reaggregation in water often could enhance channel-forming ability.
• AFM image 3D structures of these amyloids present in both native (soluble, non-membranous) form and when reconstituted in a lipid bilayer.
• AFM images of freshly dissolved peptides show globular features with average diameters of 1-10 nm ( Figure 4).
• Figure 4 Based on their size distributions in the AFM images and their comparison with images of other similar peptides (Lin et al. (2001) FASEB J., 15: 2433-2444; Zhu et al.(2000) FASEB J., 14: 1244-1254; Bhatia et al.(2000) FASEB J., 14: 1233-1243; Lin et al. (1999) Biochemistry 38: 11189-11196; Rhee et al. (1998) J. Biol. Chem. 273:
• the surface of the planar lipid-bilayer patch formed by only lipid vesicles without any amyloid peptides showed no distinguished features (data not shown).
• the average roughness of these surfaces varied by ⁇ 0.1 nm.
• the four-subunit channels, and in some cases the six-subunit channels, show an overall twofold rotational symmetry. It is possible that lower-order oligomers (e.g., dimers, trimers) could form higher-order complexes (tetramers, hexamers, etc.), although we did not ascertain their presence. Occasionally, subunits seem dislocated, breaking the symmetry of arrangement of the subunits. Pore sizes were smallest ( ⁇ 1 nm) for —synuclein and ABri and larger ( ⁇ 2 nm) for A ⁇ (l-40), ADan, amylin and SAA.
• the subunit variation is also consistent with the multiple electrical conductances observed in this work ( Figure 3) as well as others (Lin et al. (2001) FASEB J., 15: 2433-2444; Lin et al. (1999) Biochemistry 38: 11189-11196; Rhee et al. (1998) J. Biol. Chem. 273: 13379-13382; Kawahara et al. (2000) /. Biol. Chem. 275: 14077- 14083; Arispe et al. (1993) Proc. Natl. Acad. ScL USA 90: 10573-10577).
• bilayer membranes have limited ability for refolding-restructuring when incubated with amyloids and are inappropriate for ion channel reconstitutions; to our knowledge, in all published studies of ion-channel reconstitutions, either in artificial membranes (as in the present work) or in vivo in cell plasma membranes, bilayer membranes were accessible to peptides from the both sides.
• Channel-forming activity also could vary with the nature of lipid and lipid mixtures (Arispe and Doh (2002) FASEB J. 16: 1526-1536; Lin and Kagan (2002) Peptides 23: 1215-1228). Nevertheless, our data show strongly that all these peptides induce ion-channel activity when reconstituted in bilayer membranes.
• Amyloid ion-channels would provide the most direct pathway for inducing pathophysiological and degenerative effects when cells encounter amyloidogenic peptides; these channels would mediate specific ion transport (Lin et al. (2001) FASEB J., 15: 2433-2444; Lin et al. (1999) Biochemistry 38: 11189-11196; Rhee et al. (1998) J. Biol. Chem. 273: 13379-13382; Kawahara et al. (2000) J. Biol. Chem. 275: 14077-
• amyloid molecules can form stable small oligomers at physiological concentrations (low nanomolar) as well as up to micromolar levels.
• the production, oligomerization, and degradation of these amyloids is a dynamic process.
• soluble amyloids are bound to various amyloid-binding proteins and are usually cleared from cerebrospinal fluid into the bloodstream, most likely via receptor transport mechanisms across the blood-brain barrier. In the diseased brain, the level of soluble amyloids is significantly elevated.
• Nanopores were produced in microfabricated silicon membranes by electron beam lithography as well as by using a finely focused ion beam. Thermal oxide was used to shrink pore sizes, if necessary and to create an insulating surface. The chips with well defined pores were easily mounted on a double chamber plastic cells recording system allowing for controlling the buffer conditions both above and below the window. The double chamber system allowed using an AFM tip as one electrode and inserting a platinum wire as the second electrode under the membrane window, in order to measure conductance across lipid bilayers that are suspended over the pores.
• Atomic force imaging and stiffness measurement and electrical capacitance measurement indicate the feasibility of supporting lipid bilayer over well defined nanopores.
• On-line addition of gramicidin, an ion channel forming peptide resulted in characteristic ionic conductance measured using IV curve measurements.
• This system is ideally suited for direct 3D structure-function study of channel conformation.
• nanopores in silicon membranes that can be used for supported bilayer study with two fluid compartments one each below and above the channels, respectively. These silicon chips with nanopores support reconstituted lipid bilayers.
• the lipid bilayers are stiff enough to allowing their imaging with AFM. Using conducting AFM tips, electrical current was recorded for ion channels formed in the lipid bilayer and over the chip nanopores by online addition of Gramicidin, an ion channel forming peptide.
• AFM imaging of initial test pores produced by electron beam lithography shows pores with a diameter ranging from 50 nm to 200 nm or more.
• An example of such nanopores is shown in Figure 15.
• the large markers were used to find the patterns of interest by an optical microscope that is integrated with the AFM. After deposition of a lipid bilayer over the chip, the pores in the chip are covered by the bilayer. The larger corner markers are still visible since the bilayers collapse over such large openings, allowing for easy navigation, and as reference to where the nano pores are situated under the bilayer (Figure 15).
• Panel B shows a bilayer supported over the chip. The bilayer covers the pores in the chip and also reveals several holes in the bilayer.
• the AFM cross- sectional height measurements along those holes indicate the hole depth of -5.5 nm, the nominal thickness of a common lipid bilayer (Lin et at. (2001) FASEB J., 15: 2433-2444).
• the bilayers are sometimes strong enough to span even the 500 nm wide gap of the alignment marks.
• Figure 16 shows a situation where a bilayer is suspended over one part of an alignment mark, but ruptures in the other part. Most likely the bilayer was ruptured during its deposition and not during AFM imaging, since the remaining suspended part of the bilayer was stable for several AFM scans.
• AFM force curve measurements show the stiffness of a bilayer on the silicon nitride membrane (Figure 16 top), of a bilayer broken along the edge of an alignment mark ( Figure 16 middle), and of a bilayer suspended over the opening ( Figure 16 bottom).
• the increased amount of z-travel necessary to obtain the same cantilever deflection when the AFM tip is over the suspended bilayer vs for the bilayer on the silicon nitride substrate indicates that the bilayer is indeed suspended over the alignment mark hole.
• the indication of a slightly softer surface at the edge of the hole is most likely caused by the AFM tip contacting the edge of the bilayer from the side.
• the inset in Figure 16 shows a suspended bilayer in more detail, the imaging force (-1.5 nN) is low enough to have a stable membrane for repetitive imaging.
• pores produced by FBB in silicon windows were used.
• the pores in the chip were insulated and shrunk in size by thermal oxide deposition using plasma enhanced CVD. They do not contain the large alignment markers that would give rise to leakage current as is the case in our electron beam lithography produced pores, and only one pore is milled in each die.
• the FEB milling process is a direct one step process, eliminating the need for photo- or electron beam resist materials potentially contaminating the sample.
• the pore is imaged repeatedly using different bias voltages applied to the bottom electrode located under the silicon nitride membrane in the 135 raM KCl solution.
• the height image does not show the pore depth and size properly, and the pores appear shallow. This is caused by tip induced broadening due to the large tip radius of the platinum coated tips.
• the current images show bright spots where there is a current flowing between bottom electrode and tip. The 'current spots' are larger than the pore, indicating that the water layer due to humidity near the pores is conductive enough to allow current to flow thus current is not completely limited to the on-pore area exclusively.
• lipid vesicles were deposited over to chips. After 30 minutes for a bilayer to form from vesicular fusion, the excess unadsorbed lipids and vesicles were rinsed away from the surface. Bulk electrical conductance across the supported nanopore chip and the overlying adsorbed lipid bilayer was measured using a drop of 135 mM KCl solution on top of the chip in which the AFM tip holder assembly was submerged and 135 mM KCl under the nitride membrane. . The IV curve measured was near flat and indicates a conductance across the bilayer of 0.025 nS.
• Gramicidin (a known ion channel forming peptide) was added to the solution at a concentration of 0.2 mg/ml.
• the effect of Gramicidin on conductance is shown in Figure 18, while applying a IV bias to the bottom electrode under the silicon nitride membrane.
• the conductance of a gramicidin ion channel is usually small, varies considerably, is dependent upon the solvent, and influenced considerably by the nature of the detergents. Assuming a nominal conductance of approx 10 pS, we estimate the number of functional gramicidin ion channels overlaying the nanopore in the supported chip to be approx 100. No effort was made to measure single channel conductance. Moreover, no effort was made to image individual ion channels. For simultaneous high resolution imaging and conductance measurements, one can utilize a sharp tip ⁇ e.g., a nanotube) with capability to measure local conductance in fluid through the tip apex only. We have developed such a nanotube tip (Chen et al. (2006) Appl. Phys.
• Silicon-on-Insulator (SOI) wafers As shown in Figure 19, panel A the SOI wafer consists of a device layer, buried oxide layer and handle wafer of thicknesses 0.34 ⁇ m, 0.3 ⁇ m and 300 ⁇ m, respectively. A thermal oxide of 300 A thickness followed by a 1000 A low stress LPCVD silicon nitride was deposited as masldng layers for etching silicon in a KOH solution. A photolithography step was performed to open a window in the silicon nitride and oxide layers to expose the silicon wafer for etching as shown in Figure 19, panel B.
• SOI Silicon-on-Insulator
• Wafers were then etched using 33% aqueous KOH solution at 70 0 C to produce arrays of dies 7 mm x7 mm in size, each having a pyramidal shaped opening of 200 ⁇ m x 200 ⁇ m in the center. Etching was stopped at the buried oxide layer thus leaving an oxide and Si membrane window.
• the wafer after this step is shown in Figure 19, panel C, and an SEM image of the wafer backside with the pyramidal shaped opening is shown in Figure 19, panel E.
• the LPCVD silicon nitride layer was removed in hot phosphoric acid.
• the oxide layers were stripped in buffered HF leaving a silicon membrane.
• the dies were processed using a focused ion beam (FIB International), creating one nanopore, 70-150 nm in diameter, through the Si membrane in each die.
• a 70 nm diameter pore is shown in Figure 19, panel F.
• thermal oxide of various thicknesses was grown on both the top and bottom sides of the die.
• the final structure is shown in Figure 19, panel D.
• nano pore membranes were mounted on plastic liquid cells to exchange fluid above or below the membrane. IV curves were obtained in 135 mM KCl solution. Current was measured using the conductive AFM setup with the cantilevered tip holder as an electrode on the top and a reference electrode under the pore-chip ( Figure 20). In absence of an insulating coat, when buffer was present on both sides of the chip, current is measured through the complete tip holder clip. To image current only through the AFM tip apex, conductance was measured with 135 mM KCl solution only under the pore chip and a flow of humid air was maintained over the top surface. This set up also ensured that, by allowing only a local water meniscus between tip and the sample, only the local conductance through each pore was measured.
• AFM was also used to image pores after deposition of lipid vesicles (DOPC) prepared by previously described method (Lin et al. (1999) Biochemistry, 38: 11189-
• vesicles were formed by drying lmg of DOPC lipid in a glass tube, kept in a desiccator overnight, and rehydrated in buffer with occasional sonication.
• a droplet 50 micro liter, 1 mg/ml
• ionic strength was brought back to 135 mM KCl both on top as well as below the pore chip to keep the bilayer hydrated properly, and current was measured through the tip holder.
• Gramicidin an ion channel forming peptide (dissolved in milliQ water with 135 mM KCl), was added to the buffer solution, and the current was measured as a function of time while gramicidin interacted with the bilayer.
• the key component of atomic force microscopy is the probe tip, as the resolution and reliability of AFM imaging is determined by its sharpness, shape, and the nature of materials.
• Standard commercial probes made of silicon or silicon nitride have tips of a pyramid shape, that do not allow easy access to narrow or deep structural features, and generally have a relatively blunt tip radius on the order of 10 nm.
• CNTs carbon nanotubes Due to their excellent physical and chemical properties (Dresselhaus et ah, editors Carbon Nanotubes: Synthesis, Structure, Properties, and Applications, Springer, Berlin, 2001; Bower et al. (2002) Appl. Phys.
• the catalyst particle at the nanotube probe tip (or the natural dome structure in a nanotube grown by a base growth mechanism) has a finite radius of curvature, that limits the AFM resolution.
• CNC high-aspect-ratio carbon nanocone
• EBID resist-free e-beam induced deposition
• FIG. 21 First, the top surface of the cantilever (NSC/tipless, MikroMasch, USA) was coated with a -10 nm thick Ni film by e-beam evaporation.
• the acceleration voltage was 30 kV
• the beam current was 50 pA.
• the carbon dot deposition time was varied between 8 and 30 s depending on the intended size of the dot. No special carbonaceous precursor molecules were introduced as the residual carbon-containing molecules naturally presenting in the chamber were sufficient for the EBID processing to form amorphous carbon dots on the cantilever surface.
• a single carbon dot with a selected diameter of -300 nm was deposited near the front end edge of the cantilever by the EBID as illustrated in Fig. 21, panel B.
• the carbon dot serves as a convenient etch mask for chemical etching.
• the removal of the carbon dot mask after the catalyst patterning was performed with an oxygen reactive ion etch (RIE) for 1 min, which exposed the Ni island as illustrated in Figure 21, panel C.
• RIE oxygen reactive ion etch
• the growth of the CNC probe was carried out at 700 0 C for 10-20 min using a mixture of NH 3 and C 2 H 2 gas (ratio 4:1) at 3 niTorr pressure. An applied electric field was utilized to guide the growth of the nanocone along the desired direction.
• the EBID of the carbon nanodots is a simple writing technique to directly fabricate nanoscale patterns on the substrate bypassing the use of any e-beam resist layer related steps (Broers et al. (1976) Appl. Phys. Lett. 29: 596).
• the carbon deposition is caused by the dissociation of the volatile molecules adsorbed on the substrate into a nonvolatile deposit via a high-energy focused electron.
• the EBID process can more accurately pattern the catalytic island at the desired position via in situ control under high magnification of x 10 000 or higher in the SEM.
• CNTs can be controlled.
• the size of the catalyst particles does not change during growth and the resultant CNTs are equidiameter nanotubes as shown in Figure 22, panels A and B.
• the applied voltage is increased, e.g., 550 V, the diameter of the catalyst particle on the tip can be gradually reduced due to a plasma etching -sputtering- effect, as indicated in Figsure 22, panels C and D.
• the gradually diminishing catalyst size causes the nanotube diameter to change with the growth time, resulting in a nanocone configuration and the eventual complete elimination of the catalyst particle at the CNC tip, which is the key mechanism to obtain a very sharp tip.
• FIG 23, panels A and B show the SEM images of our CNC probe (marked by an arrow) grown on a tipless cantilever.
• panel A a single CNC probe grown near the edge of a tipless cantilever is shown.
• panel B a higher magnification SEM micrograph, shows a CNC with -2.5 ⁇ m height, 200 nm base diameters, and a cone angle ⁇ 5°.
• the inset in Figure 23, panel B is an example transmission electron microscopy (TEM) image of a CNC tip, which shows the tip radius of the curvature of only a few nanometers.
• TEM transmission electron microscopy
• microstructure of the nanocone appears to be a mixture of crystalline and amorphous phases in general agreement with previous work (Chen et ⁇ /.(2004) Appl. Phys. Lett. 85: 5373; Wang et al. (2005) Relat. Mater. 14: 907).
• the CNC probe in Figure 23, panel B is made intentionally tilted by manipulating the electric field direction during CVD growth. Such a tilted probe is desir able as it compensates the operation tilt angle of the AFM cantilever so that the probe itself is close to being vertical for stable imaging.
• the tilt angle of the CNC probe is -13° with respect to the normal direction of the cantilever surface.
• the probe was operated on a continuous scan mode on Au or Cu film samples for as long as 8 h.
• the lateral resolution of the obtained AFM image was not noticeably changed as compared to the initially scanned image at time zero (data not shown).
• the fabrication of a sharp and high-aspect-ratio carbon nanocone probe that possesses desirable thermal stability and mechanical toughness has been demonstrated using resist-free patterning of catalyst nanodots and electric field guided CVD growth.
• the catalyst particle on the nano-cone tip was completely removed via time- dependent size reduction, thus leading to an extremely sharp tip, that can be used for AFM imaging and deep profile analysis.

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